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    张云康, 高兴, 汪德健, 王富军, 赵健. 重组谷胱甘肽-S-转移酶-肝素酶Ⅰ在大肠杆菌中的表达、纯化及其性质[J]. 华东理工大学学报(自然科学版), 2011, (4): 458-463.
    引用本文: 张云康, 高兴, 汪德健, 王富军, 赵健. 重组谷胱甘肽-S-转移酶-肝素酶Ⅰ在大肠杆菌中的表达、纯化及其性质[J]. 华东理工大学学报(自然科学版), 2011, (4): 458-463.
    ZHANG Yun-kang, GAO Xing, WANG De-jian, WANG Fu-jun, ZHAO Jian. Expression, Purification and Characterization of Recombinant GST-haparinaseⅠ from Flavobacterium heparinum in Escherichia coli[J]. Journal of East China University of Science and Technology, 2011, (4): 458-463.
    Citation: ZHANG Yun-kang, GAO Xing, WANG De-jian, WANG Fu-jun, ZHAO Jian. Expression, Purification and Characterization of Recombinant GST-haparinaseⅠ from Flavobacterium heparinum in Escherichia coli[J]. Journal of East China University of Science and Technology, 2011, (4): 458-463.

    重组谷胱甘肽-S-转移酶-肝素酶Ⅰ在大肠杆菌中的表达、纯化及其性质

    Expression, Purification and Characterization of Recombinant GST-haparinaseⅠ from Flavobacterium heparinum in Escherichia coli

    • 摘要: 肝素酶Ⅰ(HeparinaseⅠ, HepⅠ)是一种多糖裂解酶,可特异性裂解硫酸肝素分子中糖苷键以制备低分子质量肝素(LMWH)。由于重组肝素酶Ⅰ在大肠杆菌中表达时极易形成包涵体,将肝素酶Ⅰ基因的N端融合谷胱甘肽-S-转移酶(GST)标签,在大肠杆菌中进行低温表达。结果显示,约90%的GST-HepⅠ融合蛋白以可溶性形式表达,表达的目标蛋白粗酶经过一步亲和纯化,比活为124.7 U/mg,纯化倍数达到366.7倍,酶活回收率为31.0%;结果表明Ca2+对重组GST-Hep Ⅰ有很强的激活作用,GPC-HPLC结果显示重组GST-Hep Ⅰ与天然肝素酶 Ⅰ 裂解图谱相同。

       

      Abstract: HeparinaseⅠis an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme was highly prone to aggregation in inclusion bodies when it was expressed in Escherichia coli. In this paper, the N-terminus of heparinaseⅠgene was fused a glutathione-S-transferase (GST) tag, which expressed the fusion protein in Escherichia coli. The results showed when induced at 15 ℃, approximately 90% of the fusion protein was found to be produced in the soluble. The enzyme has a specific activity of 124.7 U/mg protein by one-step affinity chromatography, the recovery was 31.0%, and a 366.7-fold-purification was achieved. GST-Hep I was well activated by Ca2+. Results of GPC-HPLC (Gel permeation chromatography high-performance liquid chromatography) analysis of oligosaccharides of heparin degraded by GST-HepⅠ was similar to those of native Hep Ⅰ.

       

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